Technical Field
The present disclosure relates to a method and a device for nucleic acid sequencing.
Description of the Related Art
Known in the art are methods for DNA sequencing. Sequencing is fundamental for characterizing a macromolecule, for example in order to determine the order of the amino acids of a protein or the sequence of bases of a nucleic acid. Sequencing of an entire genome may enable prediction of the sequence of all the proteins that this is potentially able to produce.
A method for DNA sequencing known in the prior art envisages the use of dideoxynucleotide terminators and is known as the “Sanger method”. The Sanger method consists of three steps: preparation of the sample, sequencing reaction, and electrophoresis. In the first step of preparation of the sample, the DNA strand that is to be sequenced is subjected to PCR (Polymerase Chain Reaction) in order to amplify it, i.e., obtain a plurality of identical copies thereof. Other techniques such as recombinant DNA may also be used. In the second, sequencing reaction step, the biological sample is subjected to denaturing, primer annealing, copy of the strand, and termination. With denaturing, the DNA is separated into individual strands. During primer annealing, a primer is added to the end 3′ of one of the two strands. The primer is synthesized artificially and appropriately for the DNA sequence to be sequenced.
Four mixtures are then prepared, one for each base, added to which is DNA polymerase (step of copy of the strand), the four nucleotides (dATP, dCTP, dGTP, dTTP), and an amount of a dideoxynucleotide triphosphate (ddNTP, for example, ddATP), i.e., a nucleotide without the —OH group in position 3′ of the sugar.
The above may, however, be added by the DNA polymerase to a DNA strand being synthesized via formation of a phosphodiester bond between its 5′-phosphate and 3′-OH of the previous residue. However, since ddNTPs lack the —OH group in position 3′, the subsequent nucleotide may not be bound as occurs in natural DNA replication. For this reason, the synthesis stops at the position in which a ddNTP has been incorporated at the growing end of a DNA strand (termination step).
The new strands, each of which terminates with a ddNTP, for instance a ddATP in the example considered, have lengths that are different from one another. Once DNA polymerase encounters a T base on the template strand, it may add a dATP or a ddATP. If a dATP is added, growth of the strand continues, whereas if a ddATP is added, growth of the strand stops. This process is carried out for all four nucleotides.
Then, the strands thus synthesized are denatured and separated from the template strands. At this point, the preparation is ready for the electrophoresis step.
Electrophoresis is an electrokinetic process in which charged molecules and particles, under the influence of an electrical field, migrate in the direction of a pole that has opposite charge from that of the charged molecules. Owing to the presence of the phosphate groups, the DNA molecules are negatively charged and will thus migrate towards the positive pole (anode) if subjected to an electrical field, with a rate that depends also upon their length, as well as upon the field intensity.
Introduction of capillary electrophoresis for separation of marked fragments has enabled a considerable increase in the processing rate. There have further been developed automatic sequencers that are able to carry out multiple electrophoretic runs.
However, the use of the electrophoresis technique renders the Sanger method impractical to integrate in a portable biomedical device such as one that is obtained using MEMS technology.
New-generation sequencers, which are not based upon the Sanger method, use in-vitro amplification techniques and an array system for simultaneous sequencing of millions of DNA fragments. These improvements have enabled new platforms to reduce drastically the times and costs involved even though they require a demanding post-processing step and present limits of precision above all in counting the occurrences of repeated sequences. Further, these techniques are excessive in terms of cost and complexity for cases where it is necessary to focus sequencing on small selected parts of the genome.
Alongside the sequencing systems on a large scale, there is in fact currently felt the need for a method for target sequencing, typically of a single isolated gene, of which it is desired to know the exact sequence of the bases and possible variants. Such a method, possibly integrated with a PCR amplification system, would prove useful in the case where (for example, during a diagnostic examination) there is identified the presence of a particular gene, of which it is necessary to know the exact sequence (variant).